D 1461 OULU 2018 D 1461 UNIVERSITY OF OULU P.O. Box 8000 FI-90014 UNIVERSITY OF OULU FINLAND ACTA UNIVERSITATIS OULUENSIS ACTA UNIVERSITATIS OULUENSIS ACTA DMEDICA Anu Pasanen Anu Pasanen Anu University Lecturer Tuomo Glumoff GENETIC SUSCEPTIBILITY University Lecturer Santeri Palviainen TO CHILDHOOD Postdoctoral research fellow Sanna Taskila BRONCHIOLITIS Professor Olli Vuolteenaho University Lecturer Veli-Matti Ulvinen Planning Director Pertti Tikkanen Professor Jari Juga University Lecturer Anu Soikkeli Professor Olli Vuolteenaho UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF MEDICINE; Publications Editor Kirsti Nurkkala PEDEGO RESEARCH UNIT; MEDICAL RESEARCH CENTER OULU; ISBN 978-952-62-1898-4 (Paperback) OULU UNIVERSITY HOSPITAL ISBN 978-952-62-1899-1 (PDF) ISSN 0355-3221 (Print) ISSN 1796-2234 (Online) ACTA UNIVERSITATIS OULUENSIS D Medica 1461 ANU PASANEN GENETIC SUSCEPTIBILITY TO CHILDHOOD BRONCHIOLITIS Academic dissertation to be presented with the assent of the Doctoral Training Committee of Health and Biosciences of the University of Oulu for public defence in Auditorium F101 of the Faculty of Biochemistry and Molecular Medicine (Aapistie 7), on 25 May 2018, at 12 noon UNIVERSITY OF OULU, OULU 2018 Copyright © 2018 Acta Univ. Oul. D 1461, 2018 Supervised by Professor Mika Rämet Professor Mikko Hallman Doctor Minna Karjalainen Reviewed by Docent Liisa Myllykangas Professor Ville Peltola Opponent Professor Johanna Schleutker ISBN 978-952-62-1898-4 (Paperback) ISBN 978-952-62-1899-1 (PDF) ISSN 0355-3221 (Printed) ISSN 1796-2234 (Online) Cover Design Raimo Ahonen JUVENES PRINT TAMPERE 2018 Pasanen, Anu, Genetic susceptibility to childhood bronchiolitis. University of Oulu Graduate School; University of Oulu, Faculty of Medicine; PEDEGO Research Unit; Medical Research Center Oulu; Oulu University Hospital Acta Univ. Oul. D 1461, 2018 University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland Abstract Bronchiolitis is an infection of the small airways of the lung and is a common reason for infant hospitalizations. The most common causative pathogen is the respiratory syncytial virus (RSV). Genetic factors are thought to influence the risk of bronchiolitis, and better knowledge of bronchiolitis genetics will likely help to elucidate the disease process. Severe bronchiolitis in childhood may predispose to asthma. Therefore, an effective treatment of bronchiolitis may affect the present-day as well as lifelong respiratory health. In this project, we aimed to identify genetic loci of bronchiolitis susceptibility by a genome- wide association study (GWAS) and suitable follow-up studies, and to study a previously asthma- associated CDHR3 variant for association across five bronchiolitis populations by meta-analysis. We performed the GWAS on a Finnish-Swedish case-control population and identified several loci below the suggestive genome-wide significance level. Of these, three variants showed nominal associations in a replication population from the Netherlands. One of the loci affected KCND3 expression, and two others were intergenic variants with putative regulatory potential. In a follow-up study conducted on a GWAS sub population, we identified the NKG2D locus as a candidate of susceptibility to bronchiolitis. The genomic region encompassing NKG2D variants was reportedly associated with NKG2D mRNA and protein abundance. We validated the association between NKG2D genotypes and protein expression with flow cytometry. The association between NKG2D and bronchiolitis was supported by a Finnish replication study. The meta-analysis was performed on populations from Denmark, Finland, Sweden, Germany, and the Netherlands. A potential virus-specific role for the CDHR3 variant was detected in a population that comprised mostly RSV-negative cases. In conclusion, we identified new candidates of bronchiolitis susceptibility in GWAS and subsequent studies. We found the CDHR3 variant was a potential susceptibility factor in severe non-RSV bronchiolitis and asthma. Our preliminary results provide interesting starting points for further studies. In the future, better understanding of the disease mechanisms and the relationship of bronchiolitis and asthma could provide means to design new therapeutic options. Keywords: asthma, bronchiolitis, gene expression, genetic susceptibility, genome-wide association study, infant, lower respiratory infection, meta-analysis, RSV Pasanen, Anu, Geneettinen alttius lapsuusajan bronkioliitille. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta; PEDEGO- tutkimusyksikkö; Medical Research Center Oulu; Oulun yliopistollinen sairaala Acta Univ. Oul. D 1461, 2018 Oulun yliopisto, PL 8000, 90014 Oulun yliopisto Tiivistelmä Bronkioliitti on viruksen aiheuttama alahengitystieinfektio, joka usein johtaa pienten lasten sai- raalahoitoon. Yleisin bronkioliitin aiheuttaja lapsilla on respiratory syncytial -virus (RSV). Perintötekijöiden arvellaan altistavan bronkioliitille, joten uusi tieto altistavista geeneistä voi auttaa ymmärtämään taudin taustalla olevia biologisia mekanismeja. Lapsuusiän bronkioliitin ajatellaan voivan altistaa astmalle, joten bronkioliitin tehokas hoito voi vaikuttaa merkittävästi hengitysterveyteen myös pitkällä aikavälillä. Työssä pyrittiin selvittämään lapsuusajan bronkioliitille altistavia geneettisiä tekijöitä gen- ominlaajuisella assosiaatiokartoituksella, joka toteutettiin suomalais-ruotsalaisessa tapaus-ver- rokkiväestössä. Löydökset pyrittiin varmentamaan soveltuvilla jatkotutkimuksilla. Lisäksi tar- kastelimme astmalle altistavaa CDHR3-geenin polymorfismia viidessä eurooppalaisessa bron- kioliittikohortissa käyttäen meta-analyysia. Assosiaatiokartoituksessa havaittiin useita mahdollisia bronkioliittialttiuteen vaikuttavia gee- nikohtia. Näistä kolme sai tukea hollantilaisessa väestössä tehdyssä assosiaatioanalyysissä, jos- sa testattiin assosiaatiokartoituksen lupaavimmat löydökset. Yksi altistavista polymorfismeista vaikutti KCND3-geenin ilmentymiseen, ja kaksi muuta olivat geenien välisiä, mahdollisesti gee- ninsäätelyyn osallistuvia variantteja. Assosiaatiokartoituksen osa-analyysissä NKG2D tunnistet- tiin mahdolliseksi bronkioliitille altistavaksi geeniksi. NKG2D-immuunireseptorin alentunut ilmentyminen voi tulostemme perusteella altistaa vakavalle bronkioliitille. Meta-analyysissä, jonka tutkimuskohortit olivat peräisin Tanskasta, Suomesta, Ruotsista, Saksasta ja Hollannista, todettiin mahdollinen yhteys CDHR3-geenin polymorfismin ja muun viruksen kuin RSV:n aihe- uttaman bronkioliitin välillä. Toteutimme tässä työssä ensimmäisen genominlaajuisen bronkioliittialttiutta koskevan asso- siaatiokartoituksen. Assosiaatiokartoituksessa, sitä seuranneissa jatkotutkimuksissa ja meta-ana- lyysissä tunnistimme useita lupaavia alttiusgeenejä, mutta tuloksemme vaativat varmentamista suuremmissa tutkimusväestöissä. Asiasanat: alahengitystieinfektio, astma, bronkioliitti, geenin ilmentyminen, genominlaajuinen assosiaatiokartoitus, lapsi, meta-analyysi, perinnöllinen alttius, RSV Acknowledgements This work was carried out at the department of children and adolescents and the Medical Research Center Oulu, University of Oulu and Oulu University Hospital, during the years 2013-2018. I wish to acknowledge all the people who have contributed to this work one way or another and supported me during these years. I would like to sincerely thank the trio of my supervisors including Professor Mika Rämet, Professor Mikko Hallman, and Doctor Minna Karjalainen. My principal supervisor Mika Rämet is especially acknowledged for his insightful comments and practical advice in various situations as well as for his positive and encouraging but down-to-earth attitude. I am grateful to Mikko Hallman for giving me the opportunity to work in this group and I value his optimism, curiosity and open-mindedness towards research questions and life. I would like to thank my third supervisor and dear friend Minna Karjalainen for introducing me into this project as well as for her invaluable guidance regarding scientific work and thinking as well as encouragement and advice throughout this project. I am grateful to Professor Matti Korppi for his insightful remarks regarding bronchiolitis and asthma. I wish to sincerely thank all other co-authors for their valuable contributions to the original publications. Göran Wennergren, Emma Goksör, Louis Bont, Kuldeep Kumawat, Marja Ruotsalainen, Eija Piippo- Savolainen, Ilkka Junttila, Laura Kummola, Hennie Hodemaekers, Riny Janssen, Kirsi Nuolivirta, Tuomas Jartti, Anders Husby, Johannes Waage, Astrid Sevelsted, Jakob Stokholm, Bo Chawes, Nadja Vissing, Andrea Heinzmann, Hans Bisgaard, and Klaus Bønnelykke are warmly acknowledged. It was a pleasure to work with such professional and friendly people. I would like to thank the pre-examiners of this thesis, Professor Ville Peltonen and Adjunct Professor Liisa Myllykangas, for their constructive comments to improve the contents of this thesis. I am grateful to Professor Johanna Schleutker for agreeing to be my opponent and I look forward to our discussion. Kira Heller and Deborah Kaska are acknowledged for revising the English language of the thesis. Anna Vuolteenaho is acknowledged for revising the language of the Finnish abstract. I warmly thank all former and present members of our research group. I am grateful for all the good times and educational moments we have shared during these years. I wish to thank Maarit Haarala, Johanna Huusko, Antti Haapalainen, Heli Tiensuu,